Solution structure of the Iγ subdomain of the Mu end DNA-binding domain of phage Mu transposase

Clubb, Robert T.; Schumacher, S.; Mizuuchi, Kiyoshi; Gronenborn, Angela M.; Clore, G. Marius

doi:10.1006/jmbi.1997.1312

Cited by 32 publications

(33 citation statements)

References 32 publications

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“…These essential domains have the following functions. Domain IB is a bipartite site-specific DNA binding domain that recognizes six transposase binding sites located near the ends of the Mu genome (14,15). Domain IIIA also binds DNA and has a cryptic nuclease activity implicated in catalysis of DNA cleavage (16).…”

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confidence: 99%

Mutational Analysis of the Mu Transposase

Krementsova¹,

Giffin²,

Pincus³

et al. 1998

Journal of Biological Chemistry

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Mu transposase is a member of a protein family that includes many transposases and the retroviral integrases. These recombinases catalyze the DNA cleavage and joining reactions essential for transpositional recombination. Here we demonstrate that, consistent with structural predictions, aspartate 336 of Mu transposase is required for catalysis of both DNA cleavage and DNA joining. This residue, although located 55 rather than 35 residues NH 2 -terminal of the essential glutamate, is undoubtedly the analog of the second aspartate of the AspAsp-35-Glu motif found in other family members. The core domain of Mu transposase consists of two subdomains: the NH 2 -terminal subdomain (IIA) contains the conserved Asp-Asp-Glu motif residues, whereas the smaller COOH-terminal subdomain (IIB) contains a large positively charged region exposed on its surface. To probe the function of domain IIB, we constructed mutant proteins carrying deletion or substitution mutations within this region. The activity of the deletion proteins revealed that domains IIA and IIB can be provided by different subunits in the transposase tetramer. Substitution mutations at two pairs of exposed lysine residues within the positively charged surface of domain IIB render transposase defective in transposition at a reaction step after DNA cleavage but prior to DNA joining. The severity of this defect depends on the structure of the DNA flanking the cleavage site. Thus, these data suggest that domain IIB is involved in manipulating the DNA near the cleavage site and that this function is important during the transition between the DNA cleavage and the DNA joining steps of recombination.Transposons are genetic elements that move from one site on DNA to another by transpositional recombination. Most transposons encode a single transposase protein responsible for the DNA cleavage and DNA joining reactions required during the initial recombination steps (1, 2). Retroviral integration follows a very similar recombination pathway as transposition (3, 4), and recent studies clearly establish that the transposases and retroviral integrases are a protein family whose members contain regions of similar amino acid sequence, protein structure, and function (5, 6).The transposase of bacteriophage Mu (MuA) is one of the best characterized members of the transposase/integrase family. MuA promotes two chemical steps during recombination: 1) donor DNA cleavage, which is the endonucleolytic cleavage of the phosphodiester bond between the element and the flanking host DNA, and 2) DNA strand transfer, the coupled DNA cleavage and joining reaction that covalently links the 3Ј ends of the element DNA to the DNA at the target site (5) (reviewed in Refs.

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confidence: 99%

Mutational Analysis of the Mu Transposase

Krementsova¹,

Giffin²,

Pincus³

et al. 1998

Journal of Biological Chemistry

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“…Within a transpososome, MuA subunits use three distinct domains to contact three DNA sites on the transposon ends (13)(14)(15)(16). The cleavage site is contacted by the catalytic domain, …”

Section: Flexibility and Rigidity Within A Mua Transpososomementioning

confidence: 99%

“…NMR structures have been solved for the isolated MuA domains I␤ (15) and I␥ (14). In the domain I␥ structure, the inter-domain tether is unstructured.…”

Section: Altered Dna Spacing Is Accommodated Locally In the Transposomentioning

confidence: 99%

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Sequence and Positional Requirements for DNA Sites in a Mu Transpososome

Goldhaber-Gordon¹,

Early²,

Gray³

et al. 2002

Journal of Biological Chemistry

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Transposition of bacteriophage Mu uses two DNA cleavage sites and six transposase recognition sites, with each recognition site divided into two half-sites. The recognition sites can activate transposition of non-Mu DNA sequences if a complete set of Mu sequences is not available. We have analyzed 18 sequences from a non-Mu DNA molecule, selected in a functional assay for the ability to be transposed by MuA transposase. These sequences are remarkably diverse. Nonetheless, when viewed as a group they resemble a Mu DNA end, with a cleavage site and a single recognition site. Analysis of these "pseudo-Mu ends" indicates that most positions in the cleavage and recognition sites contribute sequence-specific information that helps drive transposition, though only the strongest contributors are apparent from mutagenesis data. The sequence analysis also suggests variability in the alignment of recognition half-sites. Transposition assays of specifically designed DNA substrates support the conclusion that the transposition machinery is flexible enough to permit variability in half-site spacing and also perhaps variability in the placement of the recognition site with respect to the cleavage site. This variability causes only local perturbations in the protein-DNA complex, as indicated by experiments in which altered and unaltered DNA substrates are paired.Some of the most fundamental of biological processes occur within nucleoprotein complexes. These processes include recombination, replication, transcription, and RNA splicing. The nucleoprotein complexes are often large and elaborate, containing features that permit sophisticated regulation. As a result, dissecting the chemistry of interactions within a complex is a challenging task, but one that is critical to understanding biological function.Transposition of bacteriophage Mu, like that of other transposons, occurs within complexes called transpososomes. Transpososomes mediate at least two sequential chemical reactions, on a pathway toward transferring the transposon DNA from one site to another. The two reactions are: (i) DNA cleavage, in which a nick is introduced precisely at the end of the transposon, on the 3Ј strand and (ii) DNA strand transfer, in which the nicked strand is joined to a separate DNA molecule called the target (1, 2). The reaction sites on the transposon DNA, or "donor DNA," are defined by specific DNA sequences, but Mu target sites are not very sequence-specific (3).Transpososomes contain multiple subunits of a transposase protein, bound to DNA sequences from both of the transposon's ends (1). These protein-DNA complexes are also called "synaptic complexes" because they bring together the two ends of the transposon DNA. The phage Mu transposase, MuA, is monomeric in solution but forms a tetramer upon binding to specific DNA recognition sites near the transposon ends (4 -6).Each end of the Mu transposon has three MuA recognition sites: R1, R2, and R3 on the right end and L1, L2, and L3 on the left, not all of which are essential for transposition...

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“…15,16 The 3′ hydroxyl groups generated by the cleavage step then attack and join to opposite strands of the target DNA in a reaction called DNA strand transfer; this step generates the strand transfer complex (STC or type II complex). [17][18][19] Although the details of the structural changes that occur in the transpososome are not yet clear, recent biochemical work, 2,20 a structural model of the transpososome based on elecrron microscopy (EM) image analysis, 21 as well as highresolution structures of individual domains of MuA, 7,21,22 provide an excellent foundation for molecular studies.…”

Section: Introductionmentioning

confidence: 99%

The Dynamic Mu Transpososome: MuB Activation Prevents Disintegration

Lemberg

Schweidenback

Baker

2007

Journal of Molecular Biology

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DNA transposases use a single active center to sequentially cleave the transposable element DNA and join this DNA to a target site. Recombination requires controlled conformational changes within the transposase to ensure that these chemically distinct steps occur at the right time and place, and that the reaction proceeds in the net forward direction. Mu transposition is catalyzed by a stable complex of MuA transposase bound to paired Mu DNA ends (a transpososome). We find that Mu transpososomes efficiently catalyze disintegration when recombination on one end of the Mu DNA is blocked. The MuB activator protein controls the integration versus disintegration equilibrium. When MuB is present, disintegration occurs slowly and transpososomes that have disintegrated catalyze subsequent rounds of recombination. In the absence of MuB, disintegration goes to completion. These results together with experiments mapping the MuA-MuB contacts during DNA joining suggest that MuB controls progression of recombination by specifically stabilizing a concerted transition to the "joining" configuration of MuA. Thus, we propose that MuB's interaction with the transpososome actively promotes coupled joining of both ends of the element DNA into the same target site and may provide a mechanism to antagonize formation of single-end transposition products.

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Solution structure of the Iγ subdomain of the Mu end DNA-binding domain of phage Mu transposase

Cited by 32 publications

References 32 publications

Mutational Analysis of the Mu Transposase

Mutational Analysis of the Mu Transposase

Sequence and Positional Requirements for DNA Sites in a Mu Transpososome

The Dynamic Mu Transpososome: MuB Activation Prevents Disintegration

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